Cancer Cell

Article

JAK2V617F-Mediated Phosphorylation of PRMT5Downregulates Its Methyltransferase Activityand Promotes MyeloproliferationFan Liu,1,4 Xinyang Zhao,1,4 Fabiana Perna,1 Lan Wang,1 Priya Koppikar,2 Omar Abdel-Wahab,2 Michael W. Harr,1

Ross L. Levine,2 Hao Xu,1 Ayalew Tefferi,3 Anthony Deblasio,1 Megan Hatlen,1 Silvia Menendez,1 and Stephen D. Nimer1,*1Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute2Human Oncology and Pathogenesis ProgramMemorial Sloan-Kettering Cancer Center, New York, NY 10065, USA3Division of Hematology, Mayo Clinic, Rochester, MN 55905, USA4These authors contributed equally to this work

*Correspondence: s-nimer@mskcc.orgDOI 10.1016/j.ccr.2010.12.020

SUMMARY

The JAK2V617F constitutively activated tyrosine kinase is found in most patients with myeloproliferativeneoplasms. While examining the interaction between JAK2 and PRMT5, an arginine methyltransferase orig-inally identified as JAK-binding protein 1, we found that JAK2V617F (and JAK2K539L) bound PRMT5 morestrongly than did wild-type JAK2. These oncogenic kinases also acquired the ability to phosphorylatePRMT5, greatly impairing its ability to methylate its histone substrates, and representing a specific gain-of-function that allows them to regulate chromatin modifications. We readily detected PRMT5 phosphoryla-tion in JAK2V617F-positive patient samples, and when we knocked down PRMT5 in human CD34+ cellsusing shRNA, we observed increased colony formation and erythroid differentiation. These results indicatethat phosphorylation of PRMT5 contributes to the mutant JAK2-induced myeloproliferative phenotype.

INTRODUCTION

The myeloproliferative neoplasms (MPNs) are stem cell disor-

ders whose proliferation is thought to be driven by activating

tyrosine kinase gene mutations, such as the BCR-ABL fusion

gene in chronicmyelogenous leukemia (CML) (De Keersmaecker

and Cools, 2006). The JAK2 kinase V617F mutation is found in

most patients with non-CML MPN (Baxter et al., 2005; James

et al., 2005; Kralovics et al., 2005; Levine et al., 2005). It is

a constitutively active kinase that can phosphorylate STAT5 in

the absence of upstream signals, confer cytokine-independent

growth to Ba/F3 cells, and induce a myeloproliferative disease

in mouse models (Akada et al., 2010; James et al., 2005; Marty

et al., 2010; Mullally et al., 2010; Xing et al., 2008). In addition

to V617F, mutations within the exon 12 region of JAK2, such

as K539L, have been observed in patients with MPN, although

they are much rarer (Pikman and Levine, 2007; Scott et al.,

Significance

The JAK2V617F mutation has been found in most cases of MPkinases acquire the ability to phosphorylate and downregulatand erythroid differentiation promoting effects of JAK2V617Fbetween oncogenic kinases and histone arginine methylationhematopoietic stem/progenitor cells. These findings provide igies for the MPNs.

C

2007). Like the V617F mutation, these mutations disrupt the

negative regulation of JAK2 and lead to constitutive kinase

activity.

The JAK2 protein associates with the cytoplasmic domains of

a number of cytokine receptors and is crucial for mediating

signals triggered by several hematopoietic growth factors,

including erythropoietin (Epo), thrombopoietin (Tpo), and granu-

locyte colony-stimulating factor (G-CSF). Murine JAK2 knockout

embryos die of severe anemia on days 11–13 in utero, demon-

strating the importance of JAK2 in hematopoietic cytokine

signaling (Neubauer et al., 1998; Parganas et al., 1998). It has

been reported by several groups that the transforming effects

of JAK2V617F require an intact FERM domain, which binds to

homodimeric type I cytokine receptors (Lu et al., 2005; Wernig

et al., 2008a). This suggests that interactions between JAK2

and cytokine receptors remain capable of regulating the biolog-

ical function of JAK2V617F.

Ns, and in this study we show that oncogenic JAK2 mutante PRMT5 activity. This contributes to the myeloproliferationand represents a gain-of-function that leads to crosstalk, altering the gene expression profile and the behavior ofnsight into the pathogenesis and possible treatment strate-

ancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc. 283

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

Upon activation, the receptor-bound JAK2 phosphorylates

specific tyrosine residues of its downstream targets, activating

cell survival/proliferation-promoting signaling pathways (Ihle

and Gilliland, 2007). Several kinase cascades are activated by

JAK2V617F, including the STAT5/BCL-XL, PI3K/AKT, and

ERK/MAPK pathways (James et al., 2005; Wang et al., 2009);

however, they may not completely account for the MPN

phenotype.

The type II arginine methyltransferase PRMT5 was first identi-

fied as JAK-binding protein 1 (JBP1) in a yeast two-hybrid assay

(Pollack et al., 1999). It mediates the symmetrical dimethylation

of arginine residues within histones H2A, H3, and H4 (Ancelin

et al., 2006; Branscombe et al., 2001; Pal et al., 2004), and meth-

ylates other cellular proteins as well, such as p53, SPT5, and

MBD2 (Jansson et al., 2008; Kwak et al., 2003; Tan and Nakielny,

2006). Together with the WD40-repeat containing MEP50

protein andwith pICln, PRMT5 forms a large 20S protein arginine

methyltransferase complex, termed the ‘‘methylosome.’’ This

complex functions in RNA processing by methylating Sm

proteins and affecting snRNP biogenesis (Chari et al., 2008;

Friesen et al., 2001, 2002; Meister and Fischer, 2002). PRMT5

has been also found in the hSWI/SNF and NURD chromatin-

remodeling complexes (Le Guezennec et al., 2006; Pal et al.,

2004), where it can exert transcriptional control on target gene

expression.

Although first identified as JAK2-binding protein, there are no

functional data linking PRMT5 with JAK2. To gain insight into

JAK2V617F-induced MPN, we investigated the in vivo interac-

tion between PRMT5 and the oncogenic mutant JAK2 kinases

(JAK2V617F and JAK2K539L), and determined how this interac-

tion contributes to the myeloproliferative phenotype that they

induce.

RESULTS

PRMT5 Interacts with JAK2V617F and JAK2K539LMore Strongly than Wild-Type JAK2First, we examined whether PRMT5 interacts with JAK2 and if

the V617F (and K539L) activating mutations in JAK2 affect this

interaction. We coexpressed FLAG-PRMT5 with HA-tagged

wild-type JAK2 and JAK2V617F, or HA-PRMT5 with nontagged

versions of the wild-type JAK2, JAK2V617F, and JAK2K539L

proteins in 293T cells, and found that whereas the wild-type

JAK2 interacts with PRMT5, both the JAK2V617F and

JAK2K539L mutants bound PRMT5 more strongly than wild-

type JAK2 (Figures 1A and 1B), demonstrating that both consti-

tutively activated forms of JAK2 have increased affinity for

PRMT5. Next, to determine whether the endogenous

JAK2V617F and PRMT5 proteins interact in leukemia cells, we

performed co-immunoprecipitation (Co-IP) assays using two

different anti-JAK2 antibodies and the JAK2V617F-positive

HEL cell line. The interaction of JAK2V617F with PRMT5 was

readily detected using either antibody (Figure 1C). Because

none of the commercially available anti-PRMT5 antibodies effi-

ciently immunoprecipitates PRMT5, we also utilized a HEL cell

line that we engineered to stably express HA-tagged PRMT5.

Using an anti-HA antibody, we could detect a robust interaction

between PRMT5 and the mutant JAK2 (Figure 1D). We

confirmed that the interaction between PRMT5 and JAK2V617F

284 Cancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc.

is stronger than the interaction between PRMT5 and wild-type

JAK2 in hematopoietic cells using Ba/F3 cell lines that stably

express the wild-type or V617F mutant JAK2 proteins. Although

these cell lines express endogenous JAK2 protein (see and

compare lanes 2 and 3 to lane 1 in Figure S1A. available online),

using an anti-JAK2 antibody for the IP, we see that the mutant

JAK2V617F pulls down significantly more endogenous PRMT5

protein than does wild-type JAK2. We also determined the

subcellular localization of the JAK2-PRMT5 interaction by

performing Co-IP experiments using cytoplasmic and nuclear

fractions of HEL cells that stably express HA-tagged PRMT5

(Figure S1C). The interaction between JAK2 and PRMT5 can

be detected in both the cytoplasmic and nuclear fractions of

these hematopoietic cells. This reflects their normal localization

in the cell because the endogenous JAK2 and PRMT5 proteins

are found in both the cytoplasmic and nuclear fractions of HEL

cells (Figure S1B).

To map the region(s) in JAK2 that interacts with PRMT5,

we constructed a series of N-terminal deletion mutants of HA-

tagged JAK2 with or without the V617F substitution, and

expressed these proteins with FLAG-tagged PRMT5 in 293T

cells (Figure 1E). Co-IP experiments showed that deletion of

the first 382 amino acids (which contain the receptor-binding

FERM domain) from JAK2V617F greatly reduced its interaction

with PRMT5. Given that the C terminus of JAK2 (808-1132)

does not bind PRMT5, this indicates that the N-terminal portion

of JAK2 is responsible for binding PRMT5. The JAK2D382WT

deletion mutant protein also binds weakly to PRMT5, suggesting

that loss of the FERMdomainmay expose other epitopes in wild-

type JAK2 that can bind PRMT5.

Oncogenic JAK2 Kinases Phosphorylate PRMT5 In VivoTo determine if JAK2 kinase can directly phosphorylate PRMT5,

we performed an in vitro kinase assay using bacterially purified

GST-PRMT5 as the substrate (Figure 2A). JAK2-dependent

phosphorylation of GST-PRMT5 was readily detected because

a JAK2 inhibitor (JAK Inhibitor I) (1 mM) completely abrogated

the phosphorylation (lane 8). We next determined if JAK2 phos-

phorylates PRMT5 in vivo, by coexpressing HA-PRMT5 with

wild-type JAK2 or the JAK2 mutants in 293T cells, and using

anti-HA immunoprecipitation followed by anti-phosphotyrosine

immunoblotting. We found that PRMT5 was phosphorylated by

the mutant JAK2V617F and JAK2K539L kinases, but not the

wild-type JAK2 kinase (Figures 2B and 2C). The phosphorylation

of PRMT5 by JAK2V617F was further confirmed using an anti-

phosphotyrosine antibody to pull down proteins from 293T cells

coexpressing JAK2V617F and FLAG-tagged PRMT5 (Figure S2).

To determine whether wild-type JAK2 can phosphorylate

PRMT5 when it is activated by signaling through the erythropoi-

etin receptor (EpoR), we transfected 293T cells with JAK2 wild-

type or V617F mutant with or without the EpoR, and added

20 U/ml of Epo to the cells for 20 min (Figure 2D). Wild-type

JAK2 was activated by the presence of Epo and its receptor

(as shown by JAK2 autophosphorylation and phosphorylation

of HA-STAT5, lane 10). However, unlike JAK2V617F, the acti-

vated wild-type JAK2 kinase did not detectably phosphorylate

PRMT5, indicating that PRMT5 phosphorylation is indeed an

acquired function of themutant JAK2 kinase. Interestingly, phos-

phorylation of PRMT5 by JAK2V617F was reduced in cells

FL.PRMT5JAK2WT.HA

JAK2V617F.HA +

+

FLAG

JAK2

FLAGTubulin

IP: HA

Input

JAK2HA-PRMT5

--+ + +-- --

WT WT V617F K539L

Input

IP: HA

JAK2

JAK2

PRMT5

-- + + +-- -- ---- -- --

Input IgG JAK2IP

JAK2

PRMT5

Input

IP: HA

HA

FLAG

FLAG

HA

JAK2 -- -- WTFLVFFL

∆N382W

T

∆N382V

F

∆N807

∆N499V

F

∆N499W

T

+ FL.PRMT51 2 3 4 5 6 7 8 9

1 2 3 41 2 3 4 5

WTFLVFFL

ΔN382WT

ΔN382VFΔN499WTΔN499VFΔN807

1 1132

383

500

808

V617F

JAK2

A B

C

E

IP Input

IP: IgG HA

JAK2

PRMT5

JAK2

PRMT5

D

(125kDa)

JAK2HA

(70kDa)

IgG HA

Figure 1. The Oncogenic JAK2 Mutants Interact More Strongly with PRMT5 than Wild-Type JAK2 and Gain the Ability to Phosphorylate

PRMT5

(A) The V617Fmutation enhances the interaction between JAK2 and PRMT5. 293T cells were transiently transfected with vectors expressing FLAG-PRMT5 alone

(lane 2) or with HA-tagged wild-type (lane 3), or V617F (lane 4) JAK2 proteins. Immunoprecipitation was performed using an anti-HA antibody and immunoblotting

with anti-FLAG or anti-JAK2 antibodies.

(B) Both the V617F and K539Lmutations enhance the PRMT5/JAK2 association. HA-tagged PRMT5was coexpressed in 293T cells with wild-type (lane 3), V617F

(lane 4), or K539L (lane 5) JAK2 proteins. Proteins were precipitated by anti-HA immunoprecipitation, and the blots were probed with antibodies specific for HA,

JAK2, or PRMT5.

(C) Endogenous interaction between PRMT5 and JAK2V617F is detected in HEL cells. Proteins were precipitated from HEL cell extracts using two different anti-

JAK2 antibodies; normal rabbit IgG was used as a control for the immunoprecipitation.

(D) Interaction between HA-PRMT5 and endogenous JAK2V617F is detected in HEL cells. Co-IP was performed using a HEL cell line stably expressing

HA-PRMT5. Proteins were precipitated by either a normal mouse IgG or anti-HA antibody. Immunoblotting was performed using antibodies specific for JAK2

and PRMT5.

(E) The N-terminal region of JAK2 interacts with PRMT5. The left diagram is of full-length JAK2 and various amino terminal-deleted JAK2 proteins with or without

the V617F mutation. Numbers indicate the relevant amino acids. Right panel shows that HA-tagged JAK2 full-length or various amino terminal-deletion mutants

were coexpressed in 293T cells with FLAG-PRMT5. JAK2 and any associated PRMT5 were pulled down using anti-HA immunoprecipitation. Membranes were

immunoblotted with an anti-HA antibody to detect JAK2 and an anti-FLAG antibody to detect PRMT5. WTFL, wild-type full-length JAK2 protein; VFFL,

JAK2V617F full-length protein.

See also Figure S1.

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

overexpressing the EpoR, suggesting that the level of EpoR

expression can affect the ability of JAK2V617F to phosphorylate

PRMT5.

We next determined whether phosphorylation of endogenous

PRMT5 by JAK2V617F occurs in JAK2V617F-positive HEL

leukemia cells. HEL cells were treated with either DMSO or

JAK Inhibitor I for 16 hr, and the phosphorylated proteins were

C

immunoprecipitated using an anti-phosphotyrosine antibody

(Figure 2E). Although PRMT5 (and STAT5) is phosphorylated

in DMSO-treated HEL cells, and the phosphorylation of

both proteins is greatly reduced by the JAK2 inhibitor, PRMT5

is not phosphorylated in the TF-1 hematopoietic cells (which

express wild-type JAK2), even when the JAK2 kinase is

activated by Epo (or GM-CSF), which clearly triggers STAT5

ancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc. 285

PRMT5

MBP

JAK2 kinase(ng)GST-PRMT5(ug)

MBP(ug)JAK inhibitor I

-- 1.25 1.251.25 0.5

--0.5 2.5

-- 2.5 -- -- -- -- ---- -- -- -- -- -- --

0.5 1.251.252.5 -- -- 2.5 0.5

B

JAK2 -- -- WT V617FHA-PRMT5 -- + + +

Input

IP: HA

JAK2

P-PRMT5

PRMT5

2.5

Input

IP: HA

JAK2

P-JAK2

P-PRMT5P-STAT5

PRMT5STAT5 (95kDa)

JAK2WTJAK2VF

EpoR

JAK2

P-PRMT5

JAK2 -- -- WT VF KL

HA-PRMT5 -- + + + + + + +

PRMT5

-- -- + -- + -- -- + -- + ---- -- +-- -- +-- +---- +-- -- -- -- + + -- -- -- + +

1 2 3 4 5 6 7 8 9 10 11HA-PRMT5 HA-STAT5

InputIP

PY99 IgG

TubulinSTAT5PRMT5

D JI1 D JI1 DSTAT5

PRMT5

Input

IP: PY99

-- Epo GM-CSF

Epo+J

I1

STAT5

PRMT5

STAT5

PRMT5

Cytokine

1 2 3 4 5 6 7 8

A

C D

E F

+

Figure 2. The Constitutively Active JAK2 Mutants Phosphorylate PRMT5 In Vivo

(A) JAK2 phosphorylates PRMT5 in vitro. In vitro kinase assays were performed using bacterial-purified GST-PRMT5 and the active JAK2 kinase. The amount of

protein in the reaction is indicated. Myelin basic protein (MBP) was used as a positive control (lane 3).

(B) PRMT5 is phosphorylated by JAK2V617F in vivo. HA-PRMT5 was cotransfected into 293T cells with an empty vector or with vectors expressing wild-type or

V617F mutant JAK2 protein. PRMT5 was precipitated by anti-HA immunoprecipitation, and phosphorylation of PRMT5 was detected using a phosphotyrosine-

specific antibody (PY350).

(C) Both JAK2V617F and JAK2K539L phosphorylate PRMT5 in vivo. 293T cells were transiently transfected with HA-PRMT5 alone or cotransfected with an

increasing concentration of JAK2 wild-type, JAK2V617F- or JAK2K539L-expressing vector. HA-PRMT5was purified by anti-HA immunoprecipitation, and phos-

phorylation of PRMT5 was detected by immunoblotting with the PY350 antibody.

(D) Activated wild-type JAK2 is unable to phosphorylate PRMT5 in vivo. 293T cells were transfected with vectors expressing the proteins indicated in the figure.

Proteins were purified by anti-HA immunoprecipitation, and phosphorylation of PRMT5 and STAT5 was detected using an anti-phosphotyrosine antibody. Input

was from 5% of the total lysate. JAK2 phosphorylation was detected using an antibody specific for phospho-JAK2.

(E) Endogenous PRMT5 is phosphorylated in HEL cells. HEL cells were treated with either DMSO or 2 mM JAK Inhibitor I for 16 hr. Phosphorylated proteins were

purified using the PY99 anti-phosphotyrosine antibody. Normal mouse IgG was used as a control for the immunoprecipitation. The precipitated STAT5 and

PRMT5 proteins were detected using anti-STAT5 and anti-PRMT5 antibodies, respectively. JI1, JAK Inhibitor I.

(F) Phosphorylated PRMT5 is not detected in TF-1 cells. TF-1 cells cultured in growth medium supplemented with 2 ng/ml of recombinant human IL-3 were

deprived of cytokine for 16 hr. The cells (5 3 106) were then treated with GM-CSF (25 ng/ml), Epo (20 U/ml), or Epo plus 2 mM of JAK Inhibitor I (JI1) for

20 min. Phosphorylated STAT5 and PRMT5 proteins were immunoprecipitated using the PY99 antibody.

See also Figure S2.

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

phosphorylation (Figure 2F). These results identify PRMT5 as

a bona fide in vivo substrate of JAK2V617F, but not activated

wild-type JAK2.

Phosphorylation of PRMT5 by JAK2V617F GreatlyImpairs Its Methyltransferase ActivityPRMT5 has been shown to methylate histones H2A, H3, and H4

in vitro and in vivo. To determine whether phosphorylation of

PRMT5 affects its enzymatic activity, we purified HA-tagged

PRMT5 protein from 293T cells engineered to express either

wild-type or mutant JAK2 and HA-PRMT5. After we confirmed

286 Cancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc.

the phosphorylation of PRMT5 by JAK2V617F, we incubated

the purified PRMT5 with [3H] S-adenosylmethionine and

recombinant histone H4 (Figure 3A) or histone H2A (Figure 3B)

in an in vitro methylation assay. Although coexpression of wild-

type JAK2 had little effect on PRMT5 methyltransferase activity

(Figure 3A, lanes 8–10), JAK2V617F significantly impaired

the ability of PRMT5 to methylate histone H4 (lanes 11–13). As

expected, coexpressionofMEP50withPRMT5greatly enhanced

its enzymatic activity (lanes 2–4). Similar results were seen for

JAK2V617F (and JAK2K539L) on histone H2A methylation (Fig-

ure 3B; data not shown).

AMethylated H4

PRMT5

H4

--

1 2 3 4 5 6 7 8 9 10 11 12 13MEP50 EV JAK2WT JAK2VF

HA-PRMT5 -- --

1 2 3 4 5 6 7 8 9

MEP50

JAK2WTJAK2VF

-- -- -- -- -- -- -- -- +-- -- -- -- -- -- + + ---- -- -- -- + + -- -- --

Methylated H2A

PRMT5

H2A(14kDa)

H2A/H4R3me

H3(18kDa)

JAK2 -- WT V617F K539L

D

B

C

Genes regulated by PRMT5Genes regulated by JAK2

E

Down:353

Up:34842

Up:528

Down:23748

+

Hours 0 1 2 4

H2AR3me

H3

P-STAT5

Tubulin

F

G

DMSOJI1 CEP70

1

H3

H2AR3me

P-PRMT5-747

PRMT5

(11kDa)

SS Starve

d

DMSOJI1 CEP70

1

P-STAT5

H2AR3me

H3

HA-PRMT5 Figure 3. Phosphorylation of PRMT5 by

JAK2V617F Impairs Its HistoneMethyltrans-

ferase Activity

(A) Coexpression of JAK2V617F, but not the wild-

type JAK2, impairs the ability of PRMT5 to meth-

ylate histone H4 in vitro. An in vitro methylation

assay was performed using HA-PRMT5 purified

from 293T cells transfected with HA-PRMT5 alone

(lanes 5–7) or cotransfected with MEP50 (lanes

2–4), wild-type JAK (lanes 8–10), or JAK2V617F

(lanes 11–13). Increasing amount of proteins (10,

15, or 20 ml) were added to each methylation

reaction with 2.5 mg recombinant H4 and 1 mCi of3H-SAM. Even loading of the proteins (PRMT5

and histone H4) was visualized using Coomassie

blue staining. EV, empty vector.

(B) In vitro methylation assays were performed

with recombinant H2A and similarly purified

HA-PRMT5. Increasing amount of purified protein

(10 or 20 ml) was added to the reaction.

(C) Overexpression of the mutant JAK2 proteins,

but not the wild-type JAK2 protein, downregulates

H2A/H4 R3 methylation. The core histones were

purified from 293T cells transfected with either

an empty vector or with vectors expressing the

JAK2 proteins indicated in the figure. Methylation

of H2A/H4 R3 was detected using a rabbit poly-

clonal antibody specific for H2A/H4 R3 symmetric

dimethylation (H2A/H4R3me2s). An anti-histone

H3 antibody was used to show the equal loading.

(D) Inhibition of JAK2 kinase activity increases H2A

R3methylation in HEL cells. HEL cells were treated

with DMSO or with the JAK2 inhibitors JAK Inhib-

itor I (1 mM) or CEP701 (0.2 mM) for 2 hr. Histone

methylation was detected in cell lysates using an

antibody specific for symmetric dimethylation of

H2A/H4 R3. JI1, JAK Inhibitor I.

(E) Inhibition of JAK2 rapidly upregulates H2A R3

methylation in HEL cells. HEL cells were treated

with TG101348 (3 mM) for 0, 1, 2, and 4 hr. JAK2

kinase inhibition was monitored by measuring

STAT5 phosphorylation using an anti-phospho-

STAT5 antibody. Phosphorylation of PRMT5 was

detected with the phospho-specific PRMT5

antibody (P-PRMT5-747). Histones were purified

by acidic extraction, and H2A R3 methylation

was detected using an anti-H2A/H4R3me2s anti-

body. The levels of histone H3 and tubulin are

shown as loading controls.

(F) JAK2 inhibitors do not change the level of H2A R3 methylation in TF-1 cells. TF-1 cells growing in medium supplemented with recombinant human IL-3 were

treated with DMSO or JAK2 inhibitors (as indicated in the figure) for 2 hr. Histone methylation was detected using an anti-H2A/H4R3me2s antibody and STAT5

phosphorylation detected using a phospho-STAT5 antibody. SS, steady state. Starved cells were deprived of IL-3 for 2 hr.

(G) Gene expression profiles were generated using Affymetrix HG133 GeneChips and CEP701 (0.5 mM)-treated or shPRMT5-treated HEL cells, versus controls

(DMSO or a lentivirus expressing a scrambled shRNA, respectively). Genes reciprocally up- or downregulated R1.5-fold in both duplicate samples from drug-

treated samples, and the shPRMT5-treated samples are compared. The top shows the number of genes that are downregulated by CEP701 treatment and

upregulated by knockdown of PRMT5. The bottom illustrates the number of genes that are upregulated by CEP701 treatment and downregulated by PRMT5

knockdown.

See also Table S1.

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

We next examined whether JAK2V617F and JAK2K539L

expression affects global H2A/H4 R3 symmetric dimethylation

levels in vivo using 293T cells transiently expressing JAK2

wild-type, JAK2V617F, or JAK2K539L. Wild-type JAK2 had no

effect on the global level of H2A/H4 R3 methylation; however,

both oncogenic JAK2 kinases nearly abolished H2A/H4 R3

methylation (Figure 3C). We next assessed histone arginine

C

methylation levels in HEL cells, in the presence or absence of

JAK Inhibitor I, CEP701, which is a JAK2 (and FLT3) inhibitor

(Hexner et al., 2008) and TG101348 (Lasho et al., 2008; Wernig

et al., 2008b), the most specific JAK2 inhibitor tested. Treatment

with all three JAK2 inhibitors markedly increased H2A R3

symmetric dimethylation in the cell (Figures 3D and 3E) (H4 R3

symmetric dimethylation is not found in HEL cells in the presence

ancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc. 287

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

or absence of the JAK2 inhibitor). To determine how rapidly JAK2

inhibition affects the level of H2A/H4 R3 methylation, we

performed a time course experiment using HEL cells treated

with TG101348 (Figure 3E). An increase in H2A/H4 R3 methyla-

tion was seen within 1 hr, which peaked at the 2-hr time point.

At 4 hr, the effect on H2A/H4 R3 methylation began to reverse,

likely due to instability of the inhibitor because both STAT5 and

PRMT5 phosphorylation began to increase at the 4-hr time point.

In contrast, these JAK2 inhibitors had minimal effect on H2A/H4

R3 methylation levels in TF-1 cells, even though they blocked

STAT5 phosphorylation (Figure 3F). Thus, the oncogenic JAK2

proteins gain the ability to regulate global H2A/H4 R3 symmetric

dimethylation levels, presumably via phosphorylation of PRMT5.

Although JAK2 regulates gene transcription through the

canonical JAK2-STAT5 pathway, as well as other pathways,

our data suggest that the oncogenic JAK2 kinases (like V617F

and K539L) can also regulate gene expression via repression of

PRMT5 activity and possibly changes in the methylation of the

histone H2A and H4 tails. We performed gene expression profile

analysis onDMSO-treated versusCEP701-treatedHEL cells and

on PRMT5-directed shRNA-expressing HEL cells versus control

shRNA-expressing cells using Affymetrix HG133GeneChips.We

found 881 genes whose mRNA levels changed more than

1.5-fold in both duplicate samples of the CEP701-treated cells,

and 585 genes whose expression reproducibly changed R1.5-

fold in cellswherePRMT5wasknockeddown.Because inhibition

of JAK2 activity derepresses PRMT5 activity, we hypothesize

that genes that are reciprocally regulated between the inhibitor-

treated samples and the shPRMT5-treated samples will be regu-

lated by JAK2-induced PRMT5 phosphorylation. Indeed, we

found 90 such genes (42 upregulated by the inhibitor and down-

regulated by the shRNA, and 48 downregulated by the inhibitor

and upregulated by the shRNA), including genes involved in ribo-

somal biogenesis and autophagy (Figure 3G; Table S1).

The Major Phosphorylation Sites in PRMT5 Mapto Its N-Terminal RegionTo map the site(s) in PRMT5 phosphorylated by JAK2V617F, we

performed in vitro kinase assays and found that PRMT5 is

phosphorylated at multiple sites located between amino acids

268 and 320 (data not shown). There are six tyrosine residues

in this region (at position 280, 283, 286, 297, 304, and 307),

and we mutated the first three (M3), first four (M4), and all six

tyrosine residues (M6) to phenylalanine, and examined the

extent of PRMT5 phosphorylation in transfected 293T cells (Fig-

ure 4A). Compared to wild-type PRMT5, the M6 form of PRMT5

had markedly reduced phosphorylation when coexpressed with

JAK2V617F. The residual phosphorylation could be due to

dimerization with endogenous wild-type PRMT5, or to the pres-

ence of other phosphorylation sites within the protein. In contrast

the M3 form of PRMT5 had a similar degree of phosphorylation

as wild-type PRMT5, suggesting that the last three tyrosines

are the major sites of JAK2 phosphorylation in PRMT5.

To demonstrate that PRMT5 is phosphorylated at these tyro-

sine residues, we generated a phospho-specific anti-PRMT5

antibody (P-PRMT5-747) using a mixture of peptides containing

all combinations of phosphorylated Y297, Y304, and Y307

tyrosine residues. A dot blot assay confirmed that this antibody

recognizes the phosphorylated and not the unphosphorylated

288 Cancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc.

peptides (data not shown). To confirm that endogenous

PRMT5 is phosphorylated by JAK2V617F in HEL cells, we

treated the cells with the JAK inhibitor I, CEP701 (Figure 4B),

and TG101348 (Figure 3F) and performed several immunoblots.

These JAK2 inhibitors significantly reduced the phosphorylation

of PRMT5 and STAT5, demonstrating that endogenous PRMT5

is phosphorylated within these three tyrosine residues by

JAK2V617F. These three tyrosine residues are highly conserved

in PRMT5, from Xenopus to human (Figure S3A).

Phosphorylation of PRMT5 by JAK2V617F DisruptsIts Association with MEP50Because MEP50 markedly enhances the enzymatic activity of

PRMT5 (Figures 3A and 3B), we examined whether JAK2V617F

impairs PRMT5 activity by disrupting the PRMT5/MEP50

complex. Using 293T cells that transiently express HA-PRMT5,

and either the wild-type JAK2 or the V617F mutant protein, we

could readily co-immunoprecipitate endogenous MEP50 protein

from cells expressing wild-type JAK2 with an anti-HA antibody

(Figure 4C). However, coexpression of JAK2V617F significantly

reduced the interaction between PRMT5 andMEP50. The kinase

activity of JAK2V617F is required to disrupt the PRMT5/MEP50

association because treating the cells with JAK Inhibitor I

blocked the phosphorylation of PRMT5 and restored the interac-

tion between PRMT5 and MEP50 (Figure 4D). Expression of

JAK2V617F also disrupted the PRMT5/MEP50 interaction in

HeLa cells and U2OS cells (Figures S3B and S3C).

To determine whether phosphorylation of PRMT5 affects the

PRMT5/MEP50 complex in JAK2V617F-positive HEL cells, we

established a stable HEL cell line that expresses myc-tagged

MEP50 and purified the PRMT5/MEP50 complex using an anti-

myc antibody (Figure 4E). Although the unphosphorylated form

of PRMT5 bound to MEP50, the phosphorylated form of

PRMT5 was exclusively in the flowthrough following the immu-

noprecipitation. Thus, phosphorylation of PRMT5 by JAK2V617F

blocks its association with MEP50 in hematopoietic cells.

PRMT5 Negatively Regulates HematopoieticStem/Progenitor Cell Expansion and ErythroidDifferentiationWe examined whether decreased PRMT5 activity promotes

myeloproliferation and/or erythroid differentiation, by knocking

down PRMT5 expression in human CD34+ CB cells using

shRNA. We achieved only 60%–70% knockdown of PRMT5

mRNA but still observed a 2-fold increase in CFUs (Figure 5A).

We then overexpressed HA-tagged wild-type PRMT5, or the

M6 mutant form of PRMT5 (PRMT5M6), in CD34+ CB cells and

performed CFU assays. Consistent with the knockdown experi-

ments, PRMT5 overexpression significantly decreased colony

formation (Figure 5B), indicating that PRMT5 negatively regu-

lates progenitor cell proliferation and expansion.

We also examined whether PRMT5 activity regulates erythroid

differentiation, using in vitro liquid culture assays. We knocked

down PRMT5 or overexpressed wild-type PRMT5 or the

PRMT5M6 mutant in CD34+ CB cells and cultured the GFP+

cells in medium supporting erythroid differentiation for 1 or

2weeks. Although PRMT5 knockdown cells showed a significant

increase in CD71/Ter-119-positive cells, overexpression of

either the wild-type or the M6 mutant form of PRMT5 blocked

Input

IP: HA

JAK2

P-PRMT5

PRMT5

HA-PRMT5 -- WT M3 WT M3 M6M4M4 M6JAK2VF -- -- -- -- -- + + + +

1 2 3 4 5 6 7 8 9

A B

P-PRMT5-747

P-STAT5

PRMT5

DMSOJI1 CEP70

1

Input

IP: HA

JAK2

MEP50(50kDa)

P-PRMT5

MEP50

PRMT5

JAK2HA-PRMT5

-- -- WT V617F-- + + +

Input

IP: HA

JAK2

MEP50

MEP50

P-PRMT5

PRMT5

DMSO JI1

HA-PRMT5JAK2

-- + + + -- + + +-- -- WT VF -- -- WT VF

1 2 3 4 5 6 7 8

D

IP-M

yc-M

EP50Flo

w-thro

ugh

PRMT5

MEP50

E

C

Figure 4. Phosphorylation of PRMT5 by JAK2V617F Disrupts Its Association with MEP50

(A) Tyrosine residues within PRMT5 that are potential sites of phosphorylation (279,282,285, 296,303,306) were converted to phenylalanine residues by site-

directed mutagenesis. Wild-type or mutant HA-PRMT5 proteins were coexpressed with JAK2V617F protein in 293T cells and immunoprecipitated with an

anti-HA antibody. Phosphorylation of PRMT5 was detected using a rabbit polyclonal anti-phosphotyrosine antibody (PY350).

(B) HEL cells were treated with DMSO, JAK Inhibitor I (1 mM), or CEP701 (0.2 mM) overnight, and the phosphorylation of PRMT5 was detected by immunoblotting

using a phospho-PRMT5 antibody (P-PRMT5-747) specific for the Y297, Y304, and Y307 phosphotyrosine residues in the PRMT5 protein. The level of PRMT5

and phospho-STAT5 are also shown.

(C) Coexpression of JAK2V617F disrupts the PRMT5/MEP50 association. The HA-PRMT5/MEP50 complex was purified using an anti-HA antibody from 293T

cells expressing the proteins indicated in the figure. Phosphorylation of PRMT5 was confirmed by immunoblotting with an anti-phosphotyrosine antibody.

Coprecipitated MEP50 was detected with an anti-MEP50 antibody.

(D) The kinase activity of JAK2V617F is required to disrupt the PRMT5/MEP50 complex. DMSO or 4 mM JAK Inhibitor I (JI1) was added to the 293T cells after

transfection. Cells were collected 48 hr after transfection, and the HA-PRMT5/MEP50 complex was immunoprecipitated using an anti-HA antibody.

(E) Phosphorylated PRMT5 isolated from HEL cells does not bind MEP50. HEL cells stably expressing myc-tagged MEP50 were subjected to immunoprecipi-

tation using an anti-myc antibody. After overnight incubation, the flowthroughwas saved, and the PRMT5/MEP50 complex was eluted from the beads. Proteins in

the eluate and the flowthroughwere precipitated by TCA precipitation and resolved by SDS-PAGE gels. Themembranes were blotted using anti-PRMT5 and anti-

MEP50 antibodies, and the P-PRMT5-747 antibody, to quantify the amount of each protein in the various lanes.

See also Figure S3.

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

erythroid differentiation (Figures 5C and 5D). The ability of

PRMT5 knockdown cells to differentiate down the myeloid

lineage was also assessed; a mild reduction in CD11b-positive

cells was seen (Figure S4A).

To further determine whether downregulation of PRMT5

activity is important for JAK2V617F-induced myeloproliferation,

we overexpressed the wild-type or mutant PRMT5 (PRMT5M6)

proteins in JAK2V617F-positive CD34+ cells isolated from

therapeutic phlebotomy specimens of patients with PV and per-

formed CFU assays using 1 3 104 GFP+ cells (Figure 5E). The

control JAK2V617F-positive CD34+ cells formed predominantly

C

BFU-E colonies. However, the cells overexpressing PRMT5M6

showed no BFU-E or CFU-GEMM colonies, suggesting that

the phosphorylation-defective form of PRMT5 can abrogate

the erythroid differentiation potential of these cells.

To determine if PRMT5 is phosphorylated in JAK2V617F-

positive patient samples, we isolated CD34+ cells from thera-

peutic phlebotomy specimens taken from patients with PV

(Figure 5F), using normal CB CD34+ cells as a control. Of the

ten patient samples tested, all of the JAK2V617F- positive (or

exon 12 mutation-positive) samples had higher PRMT5 phos-

phorylation than the CD34+ CB cells or the JAK2V617F-negative

ancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc. 289

CB PV(#3)JA

K2VF+

PV(#4)JA

K2VF-

CLKO shPRMT5

pBGJR HA-PRMT5WT HA-PRMT5M6

ET(#1)

PV(#2)

HEL

JAK2V617F+

1 2 3 4 5 6

F

D

P-PRMT5-747

PRMT5

JAK2VF (exon12)+ JAK2VF-

7 8 9 10 11 12

A

E

PV(#9)

PV(#10)

PV(#8)

PV(#7)

PV(#6)

PV(#5)

pBGJR HA-PRMT5WT HA-PRMT5M6

Colon

ynum

berp

er1x

104

cells

BFUGEMMGM

pBGJR HA-PRMT5WT HA-PRMT5M6

Col

ony

num

berp

er2x

104

cells

BFUGEMMGM

LKO shPRMT5

Col

ony

num

ber p

er1x

104

BFUGEMMGM

LKO shPRMT5

Fold

chan

gevs

.con

trol

B

Figure 5. PRMT5 Activity Regulates Progenitor Cell Expansion/Differentiation in Human CD34+ Cells

(A) Knockdown of PRMT5 promotes CFU formation. Isolated human CBCD34+ cells were transduced with lentiviruses expressing a scrambled shRNA or shRNA

specific to PRMT5. GFP-positive cells were sorted 2 days after infection. Left panel shows that PRMT5 mRNA expression was detected by real-time PCR. Right

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

290 Cancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc.

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

CD34+ cells. We found less phospho-PRMT5 in two of the three

JAK2V617F-negative patient samples analyzed, compared to

the JAK2V617F+ MPN patient samples (Figure 5F, lane 6 and

11). The clinical information on these patients (including their

JAK2 mutant allele status) is summarized in Table S2. The

weak positivity in CB CD34+ cells and the high level of phos-

pho-PRMT5 in the one JAK2 wild-type patient sample suggest

that other tyrosine kinases can also phosphorylate PRMT5 in

hematopoietic cells. Computer-based programs, such as GPS

2.1 (Xue et al., 2008), indicate that the Y297, Y304, and Y307

tyrosine residues in PRMT5 are potentially phosphorylatable by

ABL1, FGFR, Src, and JAK1. In addition to examining CD34+

cells, we also examined granulocytes isolated from normal CB,

or from the phlebotomy specimens of patients with MPN, and

detected PRMT5 phosphorylation (Figure S4B).

DISCUSSION

We have determined that oncogenic mutations within the JAK2

tyrosine kinase (V617F and K539L) enhance its interaction with

PRMT5, leading to PRMT5 phosphorylation in vivo. Although

both the wild-type and mutant forms of JAK2 proteins interact

with PRMT5, phosphorylation of PRMT5 is a ‘‘gain-of-function’’

of the mutant JAK2 kinases, which reduces PRMT5methyltrans-

ferase activity and decreases global histone H2A/H4 R3

methylation.

The PRMT5-interacting region in the JAK2 protein maps to its

N terminus, making it unlikely that the mutated residues in the

JH2 domain play a critical role in this interaction. Furthermore,

it appears that the active conformation of the mutant JAK2

proteins, and not necessarily their kinase activity, allows them

to bind PRMT5 more strongly because the increased binding

persists in the presence of JAK2 kinase inhibitors (data not

shown).

We find that PRMT5 that is phosphorylated by JAK2V617F no

longer binds MEP50, even though we can detect an interaction

of MEP50 with unphosphorylated PRMT5 in HEL cells. It is

possible that much of the PRMT5:MEP50 complex that contains

unphosphorylated PRMT5 is cytoplasmic, rather than nuclear,

because we and others have shown that both proteins are found

in the nucleus and the cytoplasm, and PRMT5 is known to

panel illustrates that approximately 13 104 GFP-positive CD34+ cells were plated

tion of CFU-GM, CFU-GEMM, and BFU-E. Colonies were scored 2 weeks after th

iments with error bars indicating ±SD.

(B) Overexpression of PRMT5 inhibits colony formation. Isolated human CB CD34

together with HA-tagged wild-type PRMT5 or an HA-tagged M6 mutant form of

assays. The average results of two independent experiments are shown here. Th

(C) Knockdown of PRMT5 promotes erythroid differentiation. GFP+ CD34+ cells

erythroid differentiation for 14 days. CD71-positive/Ter-115-positive cells were d

(D) PRMT5 overexpression inhibits erythroid differentiation. HA-PRMT5 or HA

erythroid-promoting medium for 7 days; erythroid differentiation was determined

refers to the control lentiviral vector.

(E) PRMT5 overexpression inhibits colony formation in JAK2V617F-positive CD3

botomy units of patients with PV and transduced by control lentivirus or lentiviruse

in methylcellulose with cytokines supporting GM, GEMM, and BFU colony forma

(F) Phosphorylation of PRMT5 was detected in CD34+ cells isolated from patients

or from ten phlebotomy units taken from patients with MPN (lanes 2–3 and 5–12)

was detected using the P-PRMT5-747 antibody. The total amount of PRMT5 wa

cythemia vera; HEL, HEL leukemia cells.

See also Figure S4 and Table S2.

C

complex with MEP50 in both locations (Liang et al., 2007). Given

the enhanced proliferation seen following knockdown of PRMT5

in normal CD34+ cells, it would seem ‘‘easier’’ for the

JAK2V617F+ mutant cells to simply degrade, or not express

PRMT5. However, PRMT5 preferentially promotes p53-depen-

dent cell cycle arrest at the expense of p53-dependent

apoptosis (Jansson et al., 2008), and indeed, we find that knock-

ing down PRMT5 in HEL cells triggers cell death (data not

shown). Thus, JAK2V617F-expressing hematopoietic cells may

require that some PRMT5 protein be present, to complex with

MEP50, and perhaps to promote dimerization of the JAK2V617F

protein, similar to the role that the EpoR has been shown to play

(Lu et al., 2005; Wernig et al., 2008a). Given our confirmation of

the recent demonstrations that JAK2 can be found in the nucleus

(Dawson et al., 2009; Rinaldi et al., 2010), PRMT5 phosphoryla-

tion may be differentially regulated in the nucleus versus the

cytoplasm of the cell, and in hematopoietic cells versus other

cell types.

The kinase-dependent regulation of global H2A/H4 R3

symmetricmethylation reveals a link between an oncogenic tyro-

sine kinase and this particular chromatin modification. We have

also shown the dynamic nature of this regulation. Symmetric

dimethylation of histone H2A/H4 R3 has been shown to nega-

tively, as well as positively, regulate gene expression (Fabbrizio

et al., 2002; Richard et al., 2005). To gain insight into how

JAK2V617F could regulate gene expression through phosphor-

ylation of PRMT5, we performed gene expression profiling

comparing the JAK2-regulated genes (defined using a JAK2

inhibitor) with the PRMT5-regulated genes (defined using shRNA

directed against PRMT5) and found 90 genes that were recipro-

cally regulated. The recently reported interplay between histone

arginine methylation and DNA methylation (Zhao et al., 2009)

suggests that the oncogenic JAK2 kinases may not only regulate

histone arginine methylation but also DNA methylation, thereby

‘‘maintaining’’ a myeloproliferative epigenetic signature that

could be established by activation of the JAK/STAT pathway.

Recent work has shown an important role for arginine methyl-

ation in regulating hematopoiesis and leukemogenesis. PRMT1

(a type I arginine methyltransferase) has been shown to be an

essential component of MLL fusion protein-driven leukemogen-

esis (Cheung et al., 2007), and to regulate AML1(Runx1) function

in methylcellulose culture supplemented with cytokines to support the forma-

e plating. The results shown here are the averages of two independent exper-

+ cells were transduced with lentiviruses expressing either GFP alone, or GFP

PRMT5. GFP-positive cells were sorted, and 2 3 104 cells were used for CFU

e error bars indicate ±SD.

were cultured in serum-free medium supplemented with cytokines supporting

etermined by FACS analysis.

-PRMT5M6 was overexpressed in CB CD34+ cells, which were cultured in

by FACS analysis, staining for CD71 (y axis) and Glycophorin A (x axis). pBGJR

4+ cells isolated from patients with PV. CD34+ cells were isolated from phle-

s expressing HA-PRMT5 or HA-PRMT5M6. The 13 104 GFP+ cells were plated

tion. Colonies were scored 14 days after plating.

with MPN. CD34+ cells were isolated from human umbilical cord blood (lane 1)

. HEL cells were used as a positive control (lane 4). Phosphorylation of PRMT5

s also assessed by immunoblotting. ET, essential thrombocythemia; PV, poly-

ancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc. 291

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

(Zhao et al., 2008). PRMT5 has been shown to repress gamma-

globin gene expression by recruiting DNMT3A and inducing

additional repressive epigenetic marks, such as H4S1ph and

H4K20me3 (Rank et al., 2010; Zhao et al., 2009); this suggests

a role for PRMT5 in erythropoiesis (which we have demon-

strated) and hematopoiesis in general. In contrast, we found

that downregulation of PRMT5 activity (a type II arginine methyl-

transferase) promotes progenitor cell expansion and accelerates

erythroid differentiation. Forced expression of JAK2V617F

induces a PV-like disease in mouse models (Akada et al.,

2010; Marty et al., 2010; Mullally et al., 2010), and our studies

suggest that in addition to activating the STAT5 pathway, this

mutant JAK2 kinase can induce myeloproliferation and erythro-

cytosis by abrogating PRMT5 activity. Although knockdown of

PRMT5 in many cell lines, such as HEL and K562, led to

apoptosis and/or growth arrest, its downregulation in normal

CB CD34+ cells provides a proliferative signal. We too find that

PRMT5 regulates globin gene expression in HEL, CB CD34+

cells as well as in K562 cells (data not shown). However, the

distinct effects of PRMT5 knockdown on gene expression in

HEL versus CD34+ cells (data not shown) demonstrate the

importance of cell context on PRMT5 function.

Although PRMT5 is most highly phosphorylated in

JAK2V617F-positive MPN patient CD34+ cells (and in granulo-

cytes), it is also phosphorylated to some degree in JAK2V617F-

negative MPN and normal CB CD34+ cells. Overexpressing

PRMT5 (wild-type and, in particular, the M6 mutant form of

PRMT5) in JAK2V617F-positive CD34+ patient cells resulted in

a block in cell expansion and erythroid differentiation, providing

further evidence that phosphorylation and abrogation of

PRMT5 activity are important functions of this oncogenic kinase.

Nonetheless, further experiments are needed to assess the rela-

tive contribution of PRMT5phosphorylation to the various clinical

syndromes associated with the JAK2V617F mutation.

In conclusion, we have identified a ‘‘gain of function’’ for the

constitutively activated forms of JAK2 kinase (JAK2V617F and

JAK2K539L), namely phosphorylation of PRMT5, which allows

them to control transcription by regulating histone H2A and H4

arginine methylation. Given the diverse functions of PRMT5 in

the cell, further studies of the proteins methylated by PRMT5

and the pathways affected in JAK2V617F-expressing cells will

shed additional light on the molecular pathogenesis of the

MPNs.

EXPERIMENTAL PROCEDURES

Cell Culture, Transfection, and Plasmids

The human leukemia cell lines TF-1, HEL, and Ba/F3 were grown in RPMI

Media 1640 with 10% FBS (Invitrogen), supplemented with recombinant

human IL-3 (2 ng/ml) for TF-1 and Ba/F3 cells. To inhibit JAK2 activity in

HEL and TF-1 cells, cells were treated with three distinct JAK2 inhibitors:

JAK Inhibitor I (Calbiochem), CEP701 (LC Laboratories), and TG101348 at

the indicated concentrations (DMSO was used as the diluent control). In

the time course experiment, HEL cells were treated with 3 mM TG101348 for

0, 1, 2, and 4 hr. To activate JAK2 in TF-1 cells, the cells were deprived of

cytokine for 12–16 hr, and thenGM-CSF (25 ng/ml) or Epo (20U/ml) was added

to the growth medium for 20 min. Transient transfection of 293T cells was

performed using the PolyFect Transfection Reagent (QIAGEN) per the manu-

facturer’s instructions. To express the mutant or wild-type JAK2 proteins in

293T cells, the wild-type JAK2, JAK2V617F, and JAK2K539L cDNAs were

subcloned into the CMV promoter-driven pCDNA3 vector (Invitrogen) using

292 Cancer Cell 19, 283–294, February 15, 2011 ª2011 Elsevier Inc.

EcoRV and Not1 sites. FLAG-tagged or HA-tagged PRMT5 and HA-tagged

STAT5 cDNAs were also subcloned into the pCDNA3 vector through appro-

priate restriction sites. For in vitro kinase assays, the full-length PRMT5

open reading frame was subcloned into the bacterial expression vector

pSBET, which contains a glutathione S-transferase (GST) tag and a 63 Histi-

dine tag at the N and C termini of the inserted sequence, respectively.

In Vitro Kinase Assay

In vitro kinase assays were performed using commercially available JAK2

kinase (Upstate), which contains only the kinase domain (from amino acid

800 to C-terminal end), and bacterial-purified GST-PRMT5. For each reaction,

0.5 or 2.5 mg of GST-PRMT5, 0.5 or 1.25 ng of JAK2, and 5 mCi of [g-32p] ATP

were added to the kinase buffer (8 mM MOPS [pH 7.0], 0.2 mM EDTA, 10 mM

MgAc, and 0.1 mM cold ATP). Reactions were incubated at 30�C for 20 min,

and proteins resolved by gel electrophoresis.

In Vitro Methylation Assay

The methylation assay was done as described (Zhao et al., 2008). HA-PRMT5

was purified from transfected 293T cells by anti- HA immunoprecipitation. The

immobilized proteins were then incubated with 25 ml of HMT buffer (20 mMTris

[pH 8.8], 4 mM EDTA, 1 mM PMSF, 0.5 mM DTT) supplemented with 2.5 mg of

recombinant histone H4 or H2A (New England Biolabs) and 1 mCi 3H-SAM

(Amersham) at 30�C for 4 hr. The reaction was stopped by adding SDS loading

buffer, and the proteins were resolved on SDS-PAGE gels.

Microarray Analysis

RNA was isolated from HEL cells expressing control shRNA or PRMT5-

directed shRNA using the RNAeasy Plus Kit (QIAGEN), transcribed into

cDNA using random hexamer priming and Superscriptase (Invitrogen), and

then hybridized to the Affymetrix HG-U133 GeneChips. The data were

analyzed using Partek Genomic Suites, version 6.5, using a false discovery

rate (FDR) of 1% to filter our data and identify differentially expressed genes.

Duplicate, independent samples were prepared for each condition.

Isolation of Granulocytes and CD34+ Cells from Patient

Samples and/or Cord Blood

Granulocytes and mononuclear cells were isolated from human umbilical cord

blood by Ficoll-Hypaque Plus (Sigma Chemical) density centrifugation. CD34+

cells were then purified from the mononuclear cells by positive selection using

a Midi MACS separator, LS+ column, and CD34 Micro Bead Kit (Miltenyi

Biotec). For CFU and cell differentiation assays, CD34+ cells were cultured

in IMDM (Cellgro) containing 20% BIT 9500 (Stem Cell Technology) for 24 hr

before infection. The media were also supplemented with SCF (100 ng/ml),

FLT-3 (10 ng/ml), IL-6 (20 ng/ml), and TPO (100 ng/ml) (all purchased from

PeproTech).

For the analysis of patient samples, CD34+ cells and granulocytes were iso-

lated from phlebotomy units taken from patients with MPN treated at MSKCC,

with appropriate IRB-approved informed consent. All samples were obtained

as discarded samples during the routine clinical care of the patients, and they

were de-identified prior to analysis. To obtain enough cells for western blot-

ting, the CD34+ cells were cultured in medium supplemented with cytokines

(including SCF, IL3, IL6, and Epo) for 1 week before being subjected to

analysis. Phosphorylation of PRMT5 was detected using the P-PRMT5-747

antibody. The same membrane was stripped and re-probed to quantify the

total amount of PRMT5.

Generation of Lentiviruses and Infection of Primary

Hematopoietic CD34+ Cells

shRNA targeting PRMT5 or a scrambled shRNA was cloned into the

pLKO.1-GFP lentiviral vector. HA-PRMT5 WT or M6 cDNAs were cloned into

the lentiviral vector pBGJR (modified by replacing the CMV promoter with

the EF1a promoter). Viruses were produced by transfecting pLKO.1-GFP or

pBGJR vector with helper plasmids into 293T cells, according to standard

protocols. After 24 hr of growth, CD34+ cells were infected with high-titer

concentrated lentiviral suspensions in the presence of 8 mg/ml Polybrene

(Aldrich). GFP-positive cells were sorted 2 days after infection.

Cancer Cell

JAK2V617F Regulates PRMT5 Activity

CFU Assay

The 1 3 104 or 2 3 104 transduced GFP-positive CD34+ cells isolated from

patients with ET or PV were plated (in duplicate) in methylcellulose supple-

mented with Epo (5 IU/ml), SCF (50 ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml),

G-CSF (20 ng/ml), andGM-CSF (20 ng/ml). BFU-E, CFU-GM, andCFU-GEMM

colonies were scored 14 days after seeding.

In Vitro Differentiation Assay

GFP-positive CD34+ CB cells were cultured in 20% BIT in IMDM medium

with different cytokines that support erythroid or myeloid cell differentiation

for 7 or 14 days. The erythroid culture was supplemented with Epo (6 IU/ml)

and SCF (100 ng/ml), and the myeloid culture with SCF (100 ng/ml), FLT-3

(10 ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml), GM-CSF (20 ng/ml), and G-CSF

(20 ng/ml). Cells were collected and stained with CD71-APC (BD PharMingen)

andGlycophorin A-PE (Invitrogen) tomonitor erythroid differentiation, and with

CD11b-APC (BD PharMingen) to monitor myeloid differentiation.

ACCESSION NUMBERS

Coordinates have been deposited in the NCBI GEO with accession code

GSE25725.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

four figures, and two tables and can be found with this article online at

doi:10.1016/j.ccr.2010.12.020.

ACKNOWLEDGMENTS

We thankMinkui Luo and themembers of the S.D.N. laboratory for reading this

manuscript and providing thoughtful suggestions and comments. We thank

Erica Chuang for assistance in preparing this manuscript. This work was

supported by a Leukemia Lymphoma Society SCOR Grant (S.D.N.), a Starr

Foundation Award (S.D.N.), a Clinical Scholar Grant (awarded to F.L.) and

R01-CA151949 (to R.L.L.). The authors have no conflicts of interest or financial

disclosures to declare.

Received: August 3, 2010

Revised: November 10, 2010

Accepted: December 4, 2010

Published: February 14, 2011

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